Methods of diagnosing multidrug resistant tuberculosis

ABSTRACT

The invention relates to the discovery that a putative gene of Mycobacterium tuberculosis with no previously identified function is responsible for the ability of the bacterium to activate thioamide drugs. Since  M. tuberculosis  has a low rate of synonymous mutations, all mutations in this gene, identified as Rv3854c and now termed “EtaA,” are expected to inhibit the ability of a bacterium with the mutation to activate a thioamide or thiocarbonyl drug. Thus, detecting a bacterium with a mutation in this gene indicates that the bacterium is resistant to treatment with thioamides.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication Ser. No. 60/214,187, filed Jun. 26, 2000, the contents ofwhich are incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

[0002] The World Health Organization (“WHO”) estimates that as much asone-third of the world's population is infected with tuberculosis. In1998, the latest year for which estimates are available, Mycobacteriumtuberculosis (“MTb”) infected 7.25 million people and resulted in 2.9million fatalities (Farmer, P. et al., Int J Tuberc Lung Dis 2:869(1998)). Underlying these statistics is an emerging epidemic of multipledrug-resistant (“MDR”) tuberculosis that severely undermines controlefforts and is transmitted indiscriminately across national borders(Viskum, K. et al., Int J Tuberc Lung Dis 1:299 (1997); Bass, J. B. etal., Am J Respir Crit Care Med 149:1359 (1994)). Resistance to any ofthe front-line drugs generally bodes poorly for the patient, who then iscommitted to a regimen of less active “second-line” therapies. Wheremultidrug resistance is suspected, the WHO recommends that three or moredrugs be administered at the same time, to decrease the chance that theorganism will be able to develop resistance to all of the agents.

[0003] One of the most efficacious of the second-line drugs is thethioamide ethionamide (ETA) (Farmer, P. et al., supra). Like thefront-line drug, isoniazid (INH), ETA is specific for mycobacteria andis thought to exert a toxic effect on mycolic acid constituents of thecell wall of the bacillus (Rist, N. Adv Tuberc Res 10:69 (1960);Banerjee, A. et al., Science 263:227 (1994)). Current tuberculosistherapies include a large number of “prodrugs” that must bemetabolically activated to manifest their toxicity upon specificcellular targets (Barry, C. B., III et al., Biochem Pharm 59:221(2000)). The best characterized example of this is the activation of INHby the catalase-peroxidase KatG, generating a reactive form that theninactivates enzymes involved in mycolic acid biosynthesis (Slayden, R.A. et al., Microbes and Infection (2000) (in press); Heym, B. et al.,Tubercle Lung Dis 79:191 (1999)). The majority of clinically observedINH resistance is associated with the loss of this activating ability bythe bacillus (Musser, J. M., Clin Microbiol Rev 8:496 (1995)), but suchstrains typically retain their sensitivity toward ETA, suggesting thatETA activation requires a different enzyme than KatG (Rist, N., Adv.Tub. Res. 10, 69 (1960)).

[0004] In a striking achievement of molecular biology and genetics, theentire genome of a paradigm M. tuberculosis strain, H37Rv(EMBL/GenBank/DDBJ entry AL123456), was sequenced and published in 1998.(Cole, S. et al., Nature 393,537 (1998)). The genome was found tocomprise 4,411,531 base pairs, comprising 3,974 putative genes, of which3,924 were predicted to encode proteins. Each of the putative genes wasaccorded a number based on its position in the genome relative to aselected start site. The function of many of the putative genes,however, could not be determined when the genome was sequenced andpublished, and their function remains unknown today.

SUMMARY OF THE INVENTION

[0005] The present invention provides methods of determining the abilityof a Mycobacterium tuberculosis bacterium to oxidize a thioamide orthiocarbonyl, and thereby of determining the resistance of the bacteriumto a thioamide or thiocarbonyl drug or prodrug. The methods include, forexample, detecting a mutation in the EtaA gene in the bacterium, which amutation is indicative of decreased ability to oxidize a thioamide orthiocarbonyl. The wild-type sequence of the EtaA gene is set forth inSEQ ID NO: 1. Such mutations can include frameshift, missense, andnonsense mutations, as well as single nucleotide polymorphisms (SNPs)which cause amino acid substitutions in the normal sequence encoded bythe gene. In particular, the frameshift mutations can include, forexample, a deletion at position 65 of the EtaA gene sequence, anaddition at position 567, or an addition at position 811. SNPs canresult in, for example, any of the following amino acid substitutions:G43C, P51L, D58A, Y84D, T342K, and A381P.

[0006] The invention further provides methods of detecting suchmutations. These methods include, for example, amplifying the EtaA gene,or a portion thereof containing the mutation, with a set of primers toprovide an amplified product, sequencing the amplified product to obtaina sequence, and comparing the sequence of the amplified product with aknown sequence of a wild-type EtaA gene, wherein a difference betweenthe sequence of the amplified product and the sequence of the wild-typeEtaA gene indicates the presence of a mutation. The amplification can beby any of a variety of techniques, such as PCR. For example, the EtaAgene or a portion thereof can be amplified, the amplified product can besubjected to digestion by restriction enzymes, the resulting restrictionproducts can be separated to form a pattern of restriction fragmentlengths, and the pattern of restriction fragment lengths compared to apattern of restriction fragment lengths formed by subjecting thewild-type EtaA gene (or portion thereof corresponding to the portion ofthe EtaA gene amplified from the organism being screened) to the samerestriction enzymes. The amplification can be by PCR.

[0007] In preferred embodiments, the primers for amplifying the gene areselected from the group consisting of 5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′(SEQ ID NO:3); 5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′(SEQ ID NO:4), 5′ATCATCCATCCGCAGCAC 3′(SEQ IDNO:5); 5′AAGCTGCAGGTTCAACC 3′(SEQ ID NO:6); 5′GCATCGTGACGTGCTTG 3′(SEQID NO:7); 5′AAGCTGCAG GTTCAACC 3′(SEQ ID NO:8); 5′TGAACTCAGGTCGCGAAC3′(SEQ ID NO:9); 5′AACATCGTCGTGATCGG 3′(SEQ ID NO:10);5′ATTTGTTCCGTTATCCC 3′(SEQ ID NO:11); 5′AACCTAGCGTGTACATG 3′(SEQ IDNO:12); 5′TCTATTTCCCATCCAAG 3 (SEQ ID NO:13); and 5′GCCATGTCGGCTTGATTG3′(SEQ ID NO: 14). In particularly preferred embodiments, the primersare the sequences of SEQ ID NO:3 and SEQ ID NO:4. The separation of therestriction length fragments can be by gel electrophoresis. An EtaA genewith a known mutation, such as the particular mutanted EtaA genesdescribed above, can also be amplified and subjected to restrictionenzymes, and the resulting patterns compared to that of a EtaA geneobtained from a biological sample (for example, from a patient) todetermine whether the EtaA gene from the biological sample has the samemutation as that of the EtaA gene with the known mutation.

[0008] The mutations can also be detected by hybridization techniques.Conveniently, the sample nucleic acid is hybridized to a nucleic acid ofknown sequence, such as the wild-type EtaA gene or a portion thereof, orto a portion of the gene containing the mutation, under conditionssufficiently stringent that, if the reference nucleic acid is thewild-type sequence, failure of the sample to hybridize to the referencenucleic acid will indicate that it contains a mutation whereashybridization will indicate it comprises the wild-type sequence. Theconverse will be true if the reference nucleic acid comprises amutation. Either the sample nucleic acid or the reference nucleic acidcan be immobilized on a solid support.

[0009] The mutations can further be detected by detecting mutations inthe gene product. This can be accomplished, for example, by specificallybinding any of a number of antibodies, such as a single chain Fv portionof an antibody or an antibody fragment which retains antibodyrecognition, to a gene product with a mutation, wherein such binding isindicative of a mutation indicating that the organism containing themutation has decreased ability to oxidize a thioamide or thiocarbonyldrug or prodrug compared to an organism bearing a wild-type EtaA gene.Conveniently, the detection of specific binding of the antibody and thegene product can be measured in an ELISA. Mutations can also be detectedby mass spectrometry. In another embodiment, the mutation is detected byculturing the organism in the presence of ethionamide and testing forthe presence or absence of (2-ethyl-pyridin-4-yl)methanol, wherein theabsence of (2-ethyl-pyridin-4-yl)methanol indicates that the bacteriumhas a mutation which is indicative of decreased ability to oxidize athioamide. Conveniently, the ethionamide may be radiolabeled.

[0010] The invention further provides methods for screening anindividual with tuberculosis for the presence of a M. tuberculosisbacterium resistant to treatment with a thioamide or a thiocarbonyldrug, comprising obtaining a biological sample containing the bacteriumand detecting a mutation in an EtaA gene in the bacterium, whereindetecting the presence of a mutation is indicative the bacterium isresistant to treatment by a thioamide or a thiocarbonyl drug or prodrug.The method can include detecting the mutation by amplification of theEtaA gene with a set of primers to obtain a sequence, sequencing theamplified product, and comparing the sequence to that of the wild-typeEtaA gene, SEQ ID NO: 1, wherein a difference between the sequence ofthe amplified product and of the sequence of the wild-type geneindicates the presence of a mutation.

[0011] The invention further provides kits for determining the abilityof an M. tuberculosis organism to oxidize a thioamide or thiocarbonyl.Such kits include a container and appropriate primers for amplifying anEtaA gene or a portion thereof, and may further comprise one or morerestriction enzymes. In preferred embodiments, the primers foramplifying the gene are selected from the group consisting of5′-GGGGTACCGACAT TACGTTGATAGCGTGGA-3′(SEQ ID NO:3); 5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′(SEQ ID NO:4), 5′ATCATCCATCCGCAGCAC 3′(SEQ IDNO:5); 5′AAGCTGCAGGTTCAACC 3′(SEQ ID NO:6); 5′GCATCGTGACGTGCTTG 3′(SEQID NO:7); 5′AAGCTGCAG GTTCAACC 3′(SEQ ID NO:8); 5′TGAACTCAGGTCGCGAAC3′(SEQ ID NO:9); 5′AACATCGTCGTGATCGG 3′(SEQ ID NO:10);5′ATTTGTTCCGTTATCCC 3′(SEQ ID NO:11); 5′AACCTAGCGTGTACATG 3′(SEQ ID NO:12); 5′TCTATTTCCCATCCAAG 3 (SEQ ID NO: 13); and 5′GCCATGTCGGCTTGATTG3′(SEQ ID NO:14). In particularly preferred embodiments, the primers arethe sequences of SEQ ID NO:3 and SEQ ID NO:4. An EtaA gene with a knownmutation can also be included as a positive control.

[0012] In other embodiments, the kits may provide materials forperforming ELISA or immunoassays to detect organisms with decreasedability to oxidize thioamides, or to detect products of thioamidemetabolism. The kits may also contain radiolabeled ethionamide to permitdetection of labeled metabolic products in the presence of an organismwhich can metabolize the drug. Moreover, the kits may contain materialsfor performing thin-layer chromatography, and may contain(2-ethyl-pyridin-4-yl)methanol for use as a positive control.Alternatively, or in addition, the kits may include an antibody thatbinds to a product of the EtaA gene or to(2-ethyl-pyridin-4-yl)methanol. The kits may also contain instructionsfor detecting mutations in the EtaA gene, such as the specific mutationsidentified above. Detection of such mutations indicates that theorganism has decreased ability to oxidize a thioamide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1. In vivo production of (2-ethyl-pyridin-4-yl)methanol (5)from ETA by whole cells of MTh. FIG. 1A. Metabolism of radiolabeled ETAby MTh. Lanes a-h correspond to sequential supernatant samples taken attimes: 0.2, 0.25, 0.75, 1.5, 5.0, 8.5, and 25 hours, respectively. Lanei represents media autooxidation following 25 hr of incubation withoutbacterial cells. These metabolites correspond to ETA S-oxide (2), ETAnitrile (3) and ETA amide (4) FIG. 1B. Cell associated radioactivitycounts graphed against time. “DPM,” disintergrations per minute. FIG.1C. Left graph. The unknown major metabolite (5) was confirmed as(2-ethyl-pyridin-4-yl)methanol by co-chromatography with a syntheticcharacterized alcohol standard. Right hand graphs. Upper panel: HPLCcontinuous radiodetector spectrum corresponds to FIG. 1A. lane i, mediacontrol. Lower panel: HPLC continuous radiodetector spectrum correspondsto FIG. 1A, lane d, time point 1.5 hr, where the UV254 trace of(2-ethyl-pyridin-4-yl)methanol is superimposed

[0014]FIG. 2. EtaA and EtaR control ETA susceptibility and metabolism.Photographs of MSm pMH29 mycobacteria clones grown on 7H11 platescontaining the indicated concentration of drugs. “Control” indicates nodrug added. “INH 12.5” indicates isoniazid was present at 12.5 μg/ml.“ETA 2.5, 12.5, and 62.5” indicate that ethionamide was present at theμg/ml indicated. Within each photograph, the vertical columns show MSmclones which were tranformed with EtaA (a); vector control (b); or EtaR(c), respectively, and spotted in 10-fold dilutions (from top tobottom).

[0015]FIG. 3. FIG. 3A. The MSm clones shown in FIG. 2 were analyzed fortheir ability to metabolize [1-¹⁴C]ETA. Lanes a-f correspond to samplestaken at times: 0, 30, 90, 180, 330 and 900 minutes, respectively.Metabolites were identified as in FIG. 1. FIG. 3B. Cell-associatedradioactivity was determined as in FIG. 1B. “DPM,” disintegrations perminute. Squares represent MSm overexpressing EtaA, circles representwild type MSm, triangles represent MSm overexpressing EtaR. FIG. 3C.Macromolecule-associated radioactivity. “DPM,” disintegrations perminute. Columns 1, 2, and 3 show counts for MSm overexpressing EtaA,wild type MSm, and MSm overexpressing EtaR, respectively.

[0016]FIG. 4. EtaA and EtaR associated mutations and cross-resistance inpatient isolates from Cape Town, South Africa. FIG. 4A. Thiacetazone (1)and thiocarlide (2). FIG. 4B. Map of mutations in EtaA found in patientisolates resistant to ETA and thiacetazone. Chromosome coordinates andgene designations are in reference to the sequenced genome of MTb strainH37Rv. The “811 ” and “65” above the arrow denote nucleotide positionswithin the gene sequence. The notations “+1 nt” and “Δ1 nt” below thearrow denote that the patient isolate was found to have a nucleotideadded or deleted, respectively, at the position indicated. The othernotations below the arrow for the EtaA gene denote, in standard singleletter code, substitutions at positions in the amino acid sequence ofthe gene product. FIG. 4C. Cross-resistance determination of patientisolates and the associated nucleotide alterations observed. Theindividual patient isolates are listed vertically in the column entitled“Strain.” The final entry in that column is a mono-resistant strain ofMTb (ATCC 35830) obtained from the American Type Culture Collection(Manassas, Va.). The next four columns set forth the observed growth ofthe isolate when cultured with the indicated drug. ETA: ethionamide, TA:thiacetazone, TC: thiocarlide, INH: isoniazid. Susceptibility to ETA isreported as follows: S: susceptible (if the culture failed to grow at2.5 μg/ml), L: low-level resistance (if weak growth was observed at 2.5μg/ml), M: moderate resistance (if strong growth was observed at 2.5μg/ml), and H: high-level resistance (if growth was observed at 10μg/ml). Susceptibility to TA/TC/INH is reported as follows: S:susceptible (if the culture failed to grow at 0.5 μg/ml), L: low-levelresistance (if weak growth was observed at 0.5 μg/ml), M: moderateresistance (if weak growth was observed at 2.0 μg/ml), and H: high-levelresistance (if strong growth was observed at concentrations greater than2.0 μg/ml). The column titled “Nucleotide” denotes the position in thenucleotide sequence of the gene at which a mutation, if any, was found.The column titled “Amino-acid” indicates whether the nucleotide mutationdenoted in the column to its left resulted in an amino acid substitutionand, if so, the particular substitution and the position of the affectedamino acid in the normal amino acid sequence of the protein encoded byEtaA.

[0017]FIG. 5. The sequence of the EtaA gene. The coding region (SEQ IDNO: 1) consists of the 1467 numbered nucleotides. Portions of theuntranslated 5′ and 3′ regions are shown.

[0018]FIG. 6. The amino acid sequence (SEQ ID NO:2) of the proteinencoded by the EtaA gene.

DETAILED DESCRIPTION Introduction

[0019] It has now been discovered that two of the putative genes of M.tuberculosis, Rv3854c and Rv3855, regulate the susceptibility of M.tuberculosis to the major second-line drug, ethionamide (“ETA”), used totreat tuberculosis. Specifically, it has now been discovered that thegene currently known as Rv3854c is a monooxygenase. Further, it has nowbeen discovered that this gene confers upon Mycobacteria the ability toactivate thioamide and thiocarbonyl drugs from their prodrug form totheir active drug form. When the tuberculosis genome was sequenced andanalyzed in 1998, the gene was considered to bear homology to abacterial monooxygenase, but was sufficiently different to be classifiedas a separate, unknown family. Moreover, its substrate was unknown.

[0020] It has now further been discovered that the gene currently knownas Rv3855 is a regulator of expression of the monooxygenase encoded byRv3854c, and can repress its expression. In recognition of the discoveryof the functions of these genes, we have renamed Rv3854c and Rv3855 asEtaA and EtaR, respectively.

[0021] It has further been discovered that mutations in the EtaA geneare diagnostic of resistance to ETA. Analysis of patient isolatesrevealed a 100% correlation between mutations in this gene andresistance to ETA. When resistance was selected for, both frameshiftmutations, consisting of the deletion or addition of a singlenucleotide, and single nucleotide polymorphisms (“SNPs”) which resultedin the substitution of one amino acid residue for another, resulted inan ETA-resistant phenotype. It has previously been recognized that M.tuberculosis has an extremely low rate of synonymous mutations; that is,the organism has few if any random mutations which do not have afunctional effect. E.g., Sreevatsan, S., et al. Proc Natl Acad Sci USA94(18):9869-74 (1997). Accordingly, it is expected that any mutation inthis gene, whether frameshift, nonsense, missense, or SNP, will resultin an ETA-resistant phenotype. The Examples show that all the mutationsstudied, including two frameshift mutations and seven SNPs, resulted inincreased resistance to ETA. By contrast, three isolates selected for byresistance to thioacetazone which were not also cross resistant to ETA,and the wild-type strain which showed an ETA-sensitive phenotype, weremutation free in the EtaA/EtaR and intergenic regions. The knowledge ofthe EtaA gene sequence and of the function of the gene permits one ofskill in the art to readily identify any particular mutation of the EtaAgene in an ETA-resistant organism.

[0022] It has further been discovered that organisms with mutations inthe EtaA gene are resistant not only to ETA, but also to two otherthioamide compounds also used as second-line drugs. Thus, mutations inthis gene reduce or eliminate the value of at least three of the drugswhich have been used in combination therapy for MDR tuberculosis. Basedon the present findings, it can also be predicted that organisms withmutations in this gene will be resistant to other thioamide- orthiocarbonyl- based therapeutic agents.

[0023] The extensive cross-resistance among these compounds predicts twooverlapping mechanisms of resistance: (a) target associated, like theresistance between INH and ETA and (b) activation-associated, like theresistance among ETA (a thioamide), thioacetazone (a thioamide), andthiocarlide (a thiocarbonyl). Such considerations complicate appropriatedrug therapy for the treatment of multidrug-resistant tuberculosis andthe discovery of the cross-resistance to these compounds provides animportant tool to help understand the resistance mechanisms operating ina single patient, which may prove vital to determining appropriatetreatment for that patient.

[0024] These discoveries permit a much more rapid determination ofwhether the particular organism infecting a patient is resistant tothese second-line agents. Detection of mutations in the EtaA gene can beused to diagnose a phenotype resistant to treatment by ETA, and theother thioamide drugs, thiacetazone and thiocarlide, used as second-lineagents. In addition, the knowledge of the pathway by which ETA ismetabolized permits diagnosis of a drug-resistant phenotype by detectingdifferences in the rate of production of end-products or intermediates.

[0025] The diagnosis of a phenotype resistant to thioamide drugs hasimportant clinical implications. M. tuberculosis tends to developresistance to drugs when used as single agents (“monotherapy”).Drug-resistant tuberculosis is therefore generally treated with at leasttwo and preferably three different agents, since it is less likely thatthe organism will be able to develop resistance to all three of theagents simultaneously. ETA is one of the most important drugsrecommended by the World Health Organization for use in the case ofmultidrug resistant (“MDR”) strains of tuberculosis. If a patient withMDR tuberculosis has a mutation of EtaA rendering the patient resistantto thioamide therapies, however, the ETA will have limited or no effect,and it will be as if the patient has been administered only one or onlytwo agents. The chance that the M. tuberculosis strain present in thepatient will develop resistance to the other agents is thus higher thanexpected and, if such resistance develops, no drugs may be left whichare capable of effectively combating the organism.

[0026] Additionally, mutations in the EtaA gene permit rapididentification of MDR organisms by PCR and other techniques, rather thanby having to culture the organisms in the presence of variousantibiotics. This is especially useful because Mycobacteria are suchslow growers that patients not infrequently die before the Mycobacteriainfecting them can be cultured and their susceptibility determined byconventional means. The rapid identification of organisms permitted bythe invention reduces this problem, and also permits more rapidmonitoring of possible nosocomial spread. Additionally, the promptconfirmation or exclusion of possible transmission allows early clinicalintervention to prevent or reduce future outbreaks of MDR-tuberculosis.

Definitions and Terms

[0027] Units, prefixes, and symbols are denoted in their SystèmeInternational de Unites (SI) accepted form. Numeric ranges are inclusiveof the numbers defining the range. Unless otherwise indicated, nucleicacids are written left to right in 5′ to 3′ orientation; amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects orembodiments of the invention, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

[0028] Residues mutated from a known sequence are designated byconvention by listing in standard single letter code the residuenormally found at a designated position in the sequence, the position inthe sequence of the residue mutated, and the residue substituted for theoriginal residue. Thus, for example, “G43C” or “G43→C” indicates that aglycine residue normally found at position 43 of the relevant sequencehas been replaced or substituted by a cysteine.

[0029] References here to “MTb” refer to Mycobacterium tuberculosis. Thesequence of the entire genome of MTh is set forth in TubercuList, foundat http://genolist.pasteur.fr/TubercuList/.

[0030] References herein to “Rv3854c” and “EtaA” are to a gene found inMTb and designated as Accession Number Rv3854c in TubercuList at the website noted above. The EtaA gene is also designated as “EthA”. As usedherein, the term “wild-type EtaA gene” and references to the EtaA geneor EthA gene without further elaboration refer to the sequence set forthin TubercuList under Accession Number Rv3854c. The gene has 1467 basepairs and has the following coordinates in the published M. tuberculosisgenome: 4326007 and 4327473. TubercuList lists the gene as encoding a489 amino acid monooxygenase with a molecular weight of 55329.2 and a pIof 8.3315. The published sequences of the EtaA (EthA) gene and of theprotein encoded by the gene are set forth as SEQ ID NO:1 and SEQ IDNO:2, respectively.

[0031] The gene described herein as “EtaR” is also designated as “EthR.”It is available in TubercuList under accession number Rv3855.

[0032] As used herein, “antibody” includes reference to animmunoglobulin molecule immunologically reactive with a particularantigen, and includes where appropriate both polyclonal and monoclonalantibodies. The term also includes genetically engineered forms such aschimeric antibodies (e.g., humanized murine antibodies), heteroconjugateantibodies (e.g., bispecific antibodies). The term particularly refersherein to recombinant single chain Fv fragments (scFv), disulfidestabilized (dsFv) Fv fragments, or pFv fragments. The term “antibody”also includes antigen binding forms of antibodies, including fragmentswith antigen-binding capability (e.g., Fab′, F(ab′)₂, Fab, Fv and rIgG.See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co.,Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed., W. H. Freeman & Co.,New York (1998). Which particular sense or senses of the term areintended will be clear in context.

[0033] An antibody immunologically reactive with a particular antigencan be generated by recombinant methods such as selection of librariesof recombinant antibodies in phage or similar vectors, see, e.g., Huseet al., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or byimmunizing an animal with the antigen or with DNA encoding the antigen.

[0034] The terms “stringent hybridization conditions” or “stringentconditions” refer to conditions under which a nucleic acid sequence willhybridize to its complement, but not to other sequences in anysignificant degree. Stringent conditions in the context of nucleic acidhybridizations are sequence dependent and are different under differentenvironmental parameters. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes, Part I,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays,” Elsevier, New York, (1993) (the entirety ofTijssen is hereby incorporated by reference). Very stringent conditionsare selected to be equal to the T_(M) point for a particular probe. Lessstringent conditions, by contrast, are those in which a nucleic acidsequence will bind to imperfectly matched sequences. Stringency can becontrolled by changing temperature, salt concentration, the presence oforganic compounds, such as formamide or DMSO, or all of these. Theeffects of changing these parameters are well known in the art. Theeffect on T_(m) of changes in the concentration of formamide, forexample, is reduced to the following equation: T_(m)=81.5+16.6 (logNa⁺)+0.41 (%G+C)−(600/oligo length)−0.63(%formamide). Reductions inT_(m) due to TMAC and the effects of changing salt concentrations arealso well known. Changes in the temperature are generally a preferredmeans of controlling stringency for convenience, ease of control, andreversibility. Exemplary stringent conditions for detecting singlenucleotide polymorphisms are set forth in numerous references, includingWinichagoon, et al. Prenat Diagn 19:428-35 (1999); Labuda et al., AnalBiochem 275:84-92 (1999); and Bradley et al., Genet Test 2:337-41(1998).

[0035] “Solid support” and “support” are used interchangeably and referto a material or group of materials having a rigid or semi-rigid surfaceor surfaces. In many embodiments, at least one surface of the solidsupport will be substantially flat, although in some embodiments it maybe desirable to physically separate synthesis regions for differentcompounds with, for example, wells, raised regions, pins, etchedtrenches, or the like. According to other embodiments, the solidsupport(s) will take the form of beads, resins, gels, microspheres, orother geometric configurations.

Detecting Mutations in the EtaA Gene

[0036] As noted in the Introduction, MTh is known to have an extremelylow rate of “synonymous” mutations; that is, MTb rarely has randommutations that do not affect the function of the organism. Thus, anymutation in the EtaA gene is expected to alter the gene sufficiently sothat the enzyme encoded by the gene has reduced ability to activate athioamide prodrug. Thus, any mutation in the EtaA gene carried by an MTbbacillus is indicative of that that organism is resistant to therapy bythioamide drugs and, in particular, to the thioamide drugs ETA,thiacetazone, and thiocarlide.

[0037] There are a number of methods known in the art for detectingmutations in a given gene. Mutations in the gene can be found directlyby amplifying the gene in a MTb of interest and comparing the sequenceof the organism's gene to that of a reference EtaA gene sequence, suchas the one set forth in TubercuList. Alternatively, one can digestsamples of the EtaA gene of the organism of interest (such as that of aMTh isolated from a patient) and of a known ETA-susceptible MTb organismwith restriction enzymes, separate the resulting fragments byelectrophoretic techniques routine in the art (such as those taught inCurrent Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, Greene Publishing Associates, Inc./John Wiley & Sons,Inc., (1994 Supplement) (“Ausubel”)), and compare the pattern of thefragments, with a difference in the pattern of the fragments of thesample compared to that of the EtaA-susceptible organism beingindicative of an impaired ability of the organism to metabolize ETA.This method, known as “restriction fragment length polymorphism,” or“RFLP,” is well known in the art.

[0038] The nature of the mutation can be determined by, for example,sequencing the gene isolated from the individual organism. If thespecific mutation found is not one already identified as resulting inimpaired ability of the enzyme expressed from the gene, the mutation canbe tested by any of a variety of standard methods to determine theeffect of the mutation. For example, the gene can be transformed into aspecies of Mycobacteria known to be somewhat resistant to Eta comparedto wild-type (H37Rv), the gene expressed, and the activity of theresulting enzyme compared for activity against the enzyme expressed byidentical cells transformed with a wild-type EtaA gene. An exemplaryassay for transforming cells and determining the activity of the EtaAenzyme is set forth in the Examples herein.

[0039] Another method known in the art is “CFLP,” or “cleavase fragmentlength polymorphism.” This method involves amplifying the gene ofinterest, here EtaA, followed by digestion with cleavase I, which cutsthe DNA at sites dependent on secondary structure. Results are resolvedon agarose gels, forming a “bar code”- like pattern which is indicativeof the particular gene. Different patterns of cleavage digestionproducts are obtained for wild-type and mutant samples. The technique issensitive enough to detect mutations as subtle as point mutations.

[0040] Single-stranded conformation polymorphism (“SSCP”)has been usedto identify a number of different drug resistant phenotypes in selectedorganisms.” Line hybridization assays permit identification of mutantforms of genes responsible for resistance after amplification ofrelevant genes by the hybridization patterns of probes to samples.Resistance can be determined, for example, by reverse hybridization lineprobe assay, or “LiPA.” Kits for assays for several genes, such asvarious mutations in the cystic fibrosis gene, are availablecommercially from Innogenetics N. V. (Zwijnaarde, Belgium). For example,in the HLA typing assay, amplified biotinylated DNA is chemicallydenatured, and the single strands are hybridized with specificoligonucleotide probes immobilized as parallel lines on membrane-basedstrips. Then, strepavidin labeled with alkaline phosphatase is added andbound to any biotinylated hybrid previously formed. Incubation with anappropriate substrate results in a precipitate, and the reactivity ofthe probes can be determined.

[0041] A further method known in the art is temperature modulationheteroduplex chromatography (“TMHC”). The method involves amplificationof the gene of interest, here the EtaA gene, followed by denaturing ofthe PCR products and then slowly cooling, to a predetermined temperaturebased on the composition of the sample. While cooling, the PCR productsrenature forming hetero and homoduplexes which are resolved from oneanother using TMHC. Conveniently, the resolution is performed using aWAVE® DNA fragment analysis system (Transgenomic, Inc., San Jose,Calif.).

[0042] In another set of embodiments, mutations in the EtaA gene aredetected by hybridizing the gene or portions thereof from a biologicalsample, such as from an individual, against a reference nucleic acid,such as the wild-type EtaA gene or one or more EtaA genes with a knownmutation (for ease of discussion herein, the reference nucleic acidswill be termed “probes” and the sample being screened the “nucleic acidof interest”). The hybridizations can be performed while either theprobes or the nucleic acids of interest are attached to solid supports,or while they are in a fluid environment.

[0043] In one set of embodiments, the hybridizations are performed on asolid support. For example, the nucleic acids of interest (or “samples”)can be spotted onto a surface. Conveniently, the spots are placed in anordered pattern, or array, and the placement of where the nucleic acidsare spotted on the array is recorded to facilitate later correlation ofresults. The probes are then hybridized to the array. Conversely, theprobes can be spotted onto the surface to form an array and the sampleshybridized to that array. In another set of embodiments, beads are usedas solid supports. Conveniently, the beads can be magnetic or made ofmaterials responsive to magnetic force, permitting the beads to be movedor separated from other materials by externally applied magnetic fields.

[0044] The composition of the solid support can be anything to whichnucleic acids can be attached. It is preferred if the attachment iscovalent. The material for the support for use in any particularinstance should be chosen so as not to interfere with the labelingsystem to be used for the probes or the nucleic acids. For example, ifthe nucleic acids are labeled with fluorescent labels, the materialchosen for the support should not be one which fluoresces at wavelengthswhich would interfere with reading the fluorescence of the labels.

[0045] Preferably, the support is of a material to which the samples andprobes bind or one which is substantially non-porous to them, so thatthe oligonucleotides remain accessible (i.e., to the probes or thesamples) at the surface of the support. Membranes porous to the nucleicacids may be used so long as the membrane can bind sufficient amounts ofnucleic acid to permit the hybridization procedures to proceed. Suitablematerials should have chemistries compatible with oligonucleotideattachment and hybridization, as well as the intended label, andinclude, but are not limited to, resins, polysaccharides, silica orsilica-based materials, glass and functionalized glass, modifiedsilicon, carbon, metals, nylon, natural and synthetic fibers, such aswool and cotton, and polymers.

[0046] In some embodiments, the solid support has reactive groups suchas carboxy- amino- or hydroxy groups to facilitate attachment of theoligonucleotides (that is, the samples or the probes). Plastics may beused if modified to accept attachment of nucleic acids oroligonucleotides (since plastic usually has innate fluorescence, the useof non-fluorescent labels is preferred for use with plastic substrates.If plastic materials are used with fluorescent labels, appropriateadjustments should be made to procedures or equipment, such as the useof color filters, to reduce any interference in detecting results due tothe fluorescence of the substrate). Polymers may include, e.g.,polystyrene, polyethylene glycol tetraphtalate, polyvinyl acetate,polyvinyl chloride, polyvinyl pyrrolidone, buty rubber, andpolycarbonate. The surface can be in the form of a bead. Means ofattaching oligonucleotides to such supports are well known in the art,and are set forth, for example, in U.S. Pat. Nos. 4,973,493 and4,569,774 and PCT International Publications WO 98/26098 and WO97/46313. See also, Pon et al., Biotechniques 6:768-775 (1988); Damba,et al., Nuc. Acids Res. 18:3813-3821 (1990).

[0047] Alternatively, the samples can be placed in separate wells orchambers and hybridized in their respective well or chambers. The arthas developed robotic equipment permitting the automated delivery ofreagents to separate reaction chambers, including “chip” andmicrofluidic techniques, which allow the amount of the reagents used perreaction to be sharply reduced. Chip and microfluidic techniques aretaught in, for example, U.S. Pat. No. 5,800,690, Orchid, “Running onParallel Lines” New Scientist, Oct. 25, 1997, McCormick, et al., Anal.Chem. 69:2626-30 (1997), and Turgeon, “The Lab of the Future on CD-ROM?”Medical Laboratory Management Report. Dec. 1997, p.1. Automatedhybridizations on chips or in a microfluidic environment arecontemplated methods of practicing the invention.

[0048] Although microfluidic environments are one embodiment of theinvention, they are not the only defined spaces suitable for performinghybridizations in a fluid environment. Other such spaces includestandard laboratory equipment, such as the wells of microtiter plates,Petri dishes, centrifuge tubes, or the like can be used.

[0049] Another method for identifying the presence of SNPs is theoligonucleotide ligation assay (“OLA”), which may conveniently becoupled with flow cytometric analysis for rapid, accurate analysis ofSNPs. See, e.g., Iannone, M. A., et al., Cytometry, 39(2):131-40 (2000);and Jinneman, K. C., et al., J. Food Prot. 62(6):682-5 (1999). PCR andOLA can be used in tandem with yet another technique, Sequence-CodedSeparation, or “SCS,” to provide specificity, sensitivity, and multiplexcapability. See, e.g., Brinson, E. C., et al., Genet Test 1(1):61-8(1997) (erratum in Genet Test 2(4): 385 (1998)).

[0050] SNPs are also detected in the art by reverse dot blotallele-specific oligonucleotide (ASO) hybridization. See, e.g.,Winichagoon, et al. Prenat Diagn 19:428-35 (1999), and Labuda et al.,Anal Biochem 275:84-92 (1999). One method asserted to be faster than ASOhybridization for detecting single base pair changes is the so-calledamplification of refractory mutation system, or “ARMS.” See, e.g.,Bradley et al., Genet Test 2:337-41 (1998).

[0051] Mass spectrometry (“MS”) can also be used to detect SNPs. Forexample, matrix-assisted laser desorption-ionization-time-of-flight(“MALDI-TOF”) MS has been shown to be adaptable to high-throughputapplications for detecting SNPs. See, e.g., Griffin, T., and Smith, L.,Trends Biotechnol 18(2):77-84 (2000). A cost effective procedure foridentifying SNPs using MS is taught by Sauer, S., et al., Nucl Acids Res28(5):E13 (March 2000).

[0052] In addition to these gene-based techniques, a variety oftechniques are available which screen for functional changes,specifically, by screening for inhibition of monooxygenases. E.g.,Crespi, C. L., et al., Med. Chem. Res. 8(7/8):457-471 (1998); Crespi, C.L., et al., Anal Biochem 248(1):188-90 (1997). The latter referenceprovides a fluorescent method for determining the IC₅₀ for a testcompound and detailed optimizations of the procedure for nine cytochromeP450 enzymes are set forth by GENTEST Corp. (Woburn, Mass.) on-line atwww.gentest.com. Modification of this procedure for the enzyme encodedby the EtaA gene, using ETA as the substrate, will be readily apparentto persons of skill in the art. In these assays, the enzyme encoded bythe wild-type EtaA gene (the “control enzyme”) is tested to determinethe IC₅₀ of ETA. The enzyme encoded by the EtaA gene of a MTh ofinterest (the “test enzyme”), such as that obtained in a biologicalsample from a person to be screened, is then tested by the sameprocedure. A difference in the IC₅₀ of the test enzyme compared to thatof the control enzyme indicates a mutation in the gene.

[0053] Mutations in the gene can also be detected by detecting mutatedforms of the protein encoded by the gene. A mutation that results in atruncated protein or one with a conformation other than that of thenormal enzyme can be expected to have epitopes which are not present onthe normal enzyme. These mutated forms of the enzyme can be used toraise antibodies. Methods of producing polyclonal and monoclonalantibodies are known to those of skill in the art. See, e.g., Coligan(1991) Current Protocols in Immunology Wiley/Greene, N.Y.; Harlow andLane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press,N.Y.; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) LangeMedical Publications, Los Altos, Calif.; Goding (1986) MonoclonalAntibodies: Principles and Practice (2d ed.) Academic Press, New York,N.Y.; Birch and Lennox, Monoclonal Antibodies: Principles andApplications, Wiley-Liss, New York, N.Y. (1995). Antibodies so raisedare generally tested by being absorbed against the normal enzyme(conveniently, the enzyme is immobilized on a column and the antibodiesrun over the column) to remove those which cross react with the form ofthe enzyme expressed by the normal EtaA gene.

[0054] In another set of embodiments, mutations in the EtaA gene can bedetected by culturing MTb of interest, such as those isolated from abiological sample from a person being screened for resistant MTh, in amedium containing ETA and detecting whether the culture medium does ordoes not contain a metabolic product indicating that the monooxygenaseencoded by the EtaA gene is functional. For example, the tests candetect the metabolic product (2-ethyl-pyridin-4-yl)-methanol, which theresults herein establish for the first time is the product of ETAmetabolism by susceptible 25 MTh. The presence of this product in theculture medium of MTh cultured with ETA indicates that the organismbeing tested is susceptible to ETA treatment; the absence of thisproduct in the medium indicates that the organism is resistant.Conveniently, a culture of a reference ETA-susceptible MTb is grown atthe same time as a control so that the presence or absence of themetabolic product in the medium of the MTb of interest can be comparedto that present in the medium from the control organism. In preferredforms, radiolabeled ETA is used and the presence of the radiolabeledproduct is detected in the test and reference cultures over a period oftime is detected. For example, if ¹⁴C-labeled ETA is added to a cultureof M. tuberculosis, the subsequent presence of ¹⁴C-labeled(2-ethyl-pyridin-4-yl)-methanol indicates that the organisms aresusceptible to ETA.

[0055] The presence of the metabolic product can be determined by any ofa number of analytic means known in the art. The Example sectiondescribes the use of several of these methods, thin-layer chromatography(TLC) high-pressure liquid chromatography(HPLC), and mass spectrometry,to identify (2-ethyl-pyridin-4-yl)-methanol as the major metabolicproduct of EtaA-encoded monooxygenase activity. Other techniques can,however, also be used to identify this metabolic product, such asraising antibodies to (2-ethyl-pyridin-4-yl)-methanol by the methodsdiscussed above and using the antibodies to quantitate the presence orabsence of (2-ethyl-pyridin-4-yl)-methanol in culture media by ELISAs.In a preferred embodiment, the determination is made by subjecting asample from the culture to TLC in which a sample known to be(2-ethyl-pyridin-4-yl)-methanol is run as a control. Where the ETA hasbeen radioactively labeled, detection of the metabolic product can be bysubjecting the TLC to autoradiography. Immunoassays can also employchemiluminescence or electroluminescence detection systems. Such systemsinclude luminol, isoluminol, acridinium phenyl esters and otheracridinium chemiluminophores such as acridinium (N-sulphonyl)carboxamides, and ruthenium salts for the detection of conventionalenzyme-labelled conjugates. These agents are typically used in ELISAs orin conjunction with a chemiluminescent substrate.

Methods for Amplification of the EtaA Gene or Portions Thereof

[0056] Some of the detection methods discussed above employamplification of the EtaA gene. The EtaA gene or desired portionsthereof can be amplified by cloning or by other in vitro methods, suchas the polymerase chain reaction (PCR), the ligase chain reaction (LCR),the transcription-based amplification system (TAS), the self-sustainedsequence replication system (SSR). These and other amplificationmethodologies are well known to persons of skill.

[0057] Examples of these techniques and instructions sufficient todirect persons of skill through cloning exercises are found in Bergerand Kimmel, Guide to Molecular Cloning Techniques, Methods in EnzymologyVol. 152, Academic Press, Inc., San Diego, Calif. (1987) (hereinafter,“Berger”); Sambrook et al., Molecular Cloning—A Laboratory Manual (2nded.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press,N.Y. (1989), (“Sambrook et al.”); Ausubel, supra; Cashion et al., U.S.Pat. No. 5,017,478; and Carr, European Patent No. 0 246 864.

[0058] Examples of techniques sufficient to direct persons of skillthrough other in vitro amplification methods are found in Berger,Sambrook, and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No.4,683,202; PCR Protocols A Guide to Methods and Applications (Innis etal. eds) Academic Press Inc. San Diego, Calif. (1990) (“Innis”); Arnheim& Levinson (Oct. 1, 1990) C&EN 36-47; J. NIH Res., 3: 81-94 (1991); Kwohet al., Proc. Natl. Acad. Sci. USA 86: 1173 (1989); Guatelli et al.,Proc. Natl. Acad. Sci. USA 87, 1874 (1990); Lomell et al. J. Clin.Chem., 35: 1826 (1989); Landegren et al., Science, 241: 1077-1080(1988); Van Brunt, Biotechnology, 8: 291-294 (1990); Wu and Wallace,Gene, 4: 560 (1989); and Barringer et al., Gene, 89: 117 (1990).

[0059] In one preferred embodiment, the MTb EtaA gene can be isolated byroutine cloning methods. The cDNA sequence of the gene can be used toprovide probes that specifically hybridize to the EtaA gene in a genomicDNA sample (Southern blot), or to the EtaA mRNA, in a total RNA sample(e.g., in a Northern blot), or to cDNA reverse-transcribed from RNA (ina Southern blot)). Once the target EtaA nucleic acid is identified(e.g., in a Southern blot), it can be isolated according to standardmethods known to those of skill in the art (see, e.g., Sambrook et al.,supra; Berger, supra, or Ausubel, supra).

[0060] In another preferred embodiment, the MTb EtaA cDNA can beisolated by amplification methods such as polymerase chain reaction(PCR). One example of amplifying the MTb EtaA gene, including theprimers used, is set forth in the Examples. Persons of skill in the artwill recognize that other sets of primers could readily be designed fromthe sequence of MTb which would likewise amplify the EtaA gene.

[0061] In a particularly preferred embodiment, the EtaA gene can beamplified using the primers 5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′(SEQ IDNO:3) and 5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′(SEQ ID NO:4)(EtaA). Many other primer sets can be selected using standard programswidely available in the art. For example, the program “Primer3” isavailable on-line atwww-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi. This program wasused to select the primer pairs noted above, using the defaultconditions. The program was also used to select the following sequencingprimers, which can be used to amplify sections of the EtaA gene forsequencing: 5′ ATCATCCATCCGCAGCAC 3′ (SEQ ID NO:5); 5′ AAGCTGCAGGTTCAACC3′ (SEQ ID NO:6); 5′ GCATCGTGACGTGCTTG 3′ (SEQ ID NO:7);5′ AAGCTGCAGGTTCAACC 3′ (SEQ ID NO:8); 5′ TGAACTCAGGTCGCGAAC 3′ (SEQ IDNO:9); 5′ AACATCGTCGTGATCGG 3′ (SEQ ID NO:10); 5′ ATTTGTTCCGTTATCCC 3′(SEQ ID NO:11); 5′ AACCTAGCGTGTACATG 3′ (SEQ ID NO:12);5′ TCTATTTCCCATCCAAG 3 (SEQ ID NO:13); and 5′ GCCATGTCGGCTTGATTG 3′ (SEQID NO:14).

Labeling of nucleic acid probes

[0062] Where the EtaA DNA or a subsequence thereof or an MRNA of suchDNA is to be used as a nucleic acid probe, it is often desirable tolabel the sequences with detectable labels. The labels may beincorporated by any of a number of means well known to those of skill inthe art. However, in a preferred embodiment, the label is simultaneouslyincorporated during the amplification step in the preparation of thesample nucleic acids. Thus, for example, polymerase chain reaction (PCR)with labeled primers or labeled nucleotides will provide a labeledamplification product. In another preferred embodiment, transcriptionamplification using a labeled nucleotide (e.g. fluorescein-labeled UTPand/or CTP) incorporates a label into the transcribed nucleic acids.

[0063] Alternatively, a label may be added directly to an originalnucleic acid sample (e.g., MRNA, polyA MRNA, cDNA, etc.) or to theamplification product after the amplification is completed. Means ofattaching labels to nucleic acids are well known to those of skill inthe art and include, for example nick translation or end-labeling (e.g.with a labeled DNA) by kinasing of the nucleic acid and subsequentattachment (ligation) of a nucleic acid linker joining the samplenucleic acid to a label (e.g., a fluorophore).

[0064] Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Useful labels in the present invention include biotin for staining withlabeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein, texas red, rhodamine, greenfluorescent protein, and the like), radiolabels (e.g., ³H, 125I, ³⁵S,¹⁴C, or ³²p), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and colorimetriclabels such as colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, latex, etc.) beads. Patents teaching the useof such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

[0065] Means of detecting such labels are well known to those of skillin the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted light. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

Kits

[0066] The invention further provides kits for determining the abilityof a M. tuberculosis bacterium to metabolize a thioamide, therebypermitting a determination of whether the bacterium is susceptible orresistant to thioamide- or thiocarbonyl- based agents. The kits can takeany of a variety of forms, such as:

[0067] a kit for performing TLC to detect the presence of(2-ethyl-pyridin-4-yl)methanol will usually provide a sample of(2-ethyl-pyridin-4-yl)methanol which can be run in parallel with theexperimental sample to provide a positive control;

[0068] a kit may provide radiolabeled ETA so that the presence orabsence of a product of EtaA metabolism can be detected. For example,the kit may provide ¹⁴C-labeled ETA so that the presence or absence oflabeled (2-ethyl-pyridin-4-yl)methanol can be detected;

[0069] a kit may provide primers for amplifying an EtaA gene or aportion thereof containing a mutation that affects the ability of thebacterium to oxidize a thioamide, such as5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′(SEQ ID NO:3) and5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′(SEQ ID NO:4), or any of theother primer pairs set forth above. It should be noted that, due to thelow synonymous rate of mutation of M. tuberculosis, it is believed thatall naturally-occurring mutations in the EtaA gene will reduce theability of the organism to oxidize a thioamide. The kit may also includeisolated EtaA genes containing one or more mutations from the wild-typesequence (SEQ ID NO: 1), or nucleic acid sequences derived from such anEtaA gene, for use as a positive control during PCR or otheramplification procedures;

[0070] a kit may provide one or more antibodies which permit the use ofELISAs or other immunoassays known in the art. Typically, the antibodieswill be raised against (2-ethyl-pyridin-4-yl)methanol, to permitdetection of whether this metabolic product is produced by a particularculture, or antibodies against the gene product of the wild-type EtaAgene, or against a gene product expressed from a missense, nonsense, orframeshift mutation of the EtaA gene.

EXAMPLES Example 1. Synthesis of 2-ethyl-[¹⁴C]thioisonicotinamide(1-[¹⁴C]ETA).

[0071] 2-ethylpyridine was converted to its N-oxide salt in almostquantitative yield using 35% hydrogen peroxide in acetic acid and thecorresponding N-oxide was subjected to a nitrating mixture of sulfuricand nitric acids to form 2-ethyl-4-nitropyridine N-oxide in 60% yield(Kucherova, et al., Zhurnal Obshchei Khimii 29:915-9 (1959). Reductionusing iron filings, hydrochloric, and acetic acid (Gutekunst and Gray,J. Am. Chem Soc., 44:1741 (1922)) allowed us to isolate2-ethyl-4-aminopyridine, which was converted to 2-ethyl-4-bromopyridinethrough the perbromide using 50% aqueous hydrobromic acid and sodiumnitrite (Kucherova et al., supra). The resulting bromide was heated withcopper cyanide in N-methylpyrrolidin-2-one to afford2-ethyl-4-[¹⁴C]cyanopyridine (Lawrie et al., J Labelled CompoundsRadiopharmaceutic 36:891-8 (1995)). [¹⁴C]-copper cyanide was obtainedfrom [¹⁴C]-sodium cyanide (Amersham Pharmacia Biotech, Inc., Piscataway,N.J. 08855) using copper (II) sulfate pentahydrate and sodium sulfite(Sunay et al., J Labelled Compounds Radiopharmaceutic 36:529-36 (1995);Meinert et al., J Labelled Compounds Radiopharmaceutic 14:893-6 (1978)).The nitrite was converted to 1-[¹⁴C]-ETA by hydrogen sulfide treatmentand the resulting thioamide was purified to 98% final radiochemicalpurity using normal phase HPLC with a preparative ADSORBOSPHERE silicacolumn (5 μ, 300×22 mm, Alltech Associates, Deerfield, Ill.) and anisochratic eluent of 90% chloroform, 10% methanol. Unlabeled ETAsynthesized using the same procedure co-chromatographed withcommercially available ETA (Sigma-Aldrich Chemical Company, Milwaukee,Wis.) and showed correct analytical data.

Example 2. Materials and Methods for Determining In vivo Metabolism of1-[¹⁴C]ETA

[0072] The indicated mycobacterial species were grown in culture to anOD650 of 1.0-1.5 and then concentrated 10-fold in middlebrook 7H9 brothmedia (DIFCO laboratories, Detroit, Mich.). The culture suspensions weretreated with 0.01 μg ml⁻¹ of [¹⁴C]-ETA (55 mCi/mmol) and sequentialculture aliquots were removed at the indicated times, filtered and flashfrozen. Samples of 2 μl were analyzed by TLC on Silica gel 60 plates (EMScience, Gibbstown, N.J. 08027) developed with 95:5 ethylacetate:ethanol. Prior to spotting radioactive samples on TLC plates asmall amount of unlabeled ETA was spotted to circumvent silica-catalyzedair oxidation of the low concentration radioactive ETA samples.

[0073] Metabolites were identified by comparison with well characterizedsynthetic standards prepared as follows: the sulfoxide was prepared byhydrogen peroxide oxidation of ETA as previously described (Walter andCurtis, Chem Ber 93:1511 (1960)). The acid was made by reflux hydrolysisof the thioamide with 30% NaOH (Aq); ¹H-NMR (CDCl₃:CD₄OD;1:1); δ1.26 t,2.83 q, 7.63 d, 7.71 s, 8.52 d; ES-MS (MH+) 152.1 m/e. The amide (4) wasmade by treating the corresponding acid chloride with ammoniumhydroxide; ¹H-NMR (CDCl₃); δ1.34 t, 2.91 q, 7.45 d, 7.55 s, 8.65 d;ES-MS (MH+) 151.2 m/e. (2-ethyl-pyridin-4-yl)-methanol (5) was made byRedAl reduction of the acid in THF; ¹H-NMR (CDCl³); δ1.28 t, 2.84 q,7.11 d, 7.19 s, 8.48 d; ¹³C-NMR (CDCl₃); 14.12, 30.54,63.92,118.73,119.64,149.25,150.60,163.89; ES-MS (MH+) 138.0m/e.

[0074] Cells from sequential culture aliquots from the metabolicconversion assays (volumes given in figure legends) were collected byfiltration onto 0.22 micron GS filter disks (Millipore, Bedford, Mass.)under vacuum on a Hoeffer apparatus and were washed twice with 0.1 mMsodium phosphate, pH 7.5, 100 mM NaCl (500 μl). The cell associatedradioactivity was measured in 4 ml of EcoscintA scintillation solution(National Diagnostics, Atlanta, Ga.). HPLC separation of the [¹⁴C]-ETAmetabolite mixture was achieved using a reverse-phase LUNA column (5 μ,C18(2), 250×4.6 mm, Phenomenex, Torrence, Calif.) with a gradient of:(0-5 min) 0% acetonitrile, 100 % water; then (5-65 min) to 70 %acetonitrile; then (65-80 min) to 100 % acetonitrile (all solventscontained 0.1 % trifluoroacetic acid). The retention time of the unknownradiolabeled major metabolite (5) utilizing continuous radiodetection(β-RAM, INUS Systems, Florida), was used to guide cold large scale ETAfeeding experiments with up to 1 liter of log phase MTh H37Rv, to whichwe fed 10 μg ml⁻¹ ETA (Sigma-Aldrich, Milwaukee, Wiss.). We HPLCisolated very small quantities of unlabeled metabolite with a similarretention time to (5), utilizing UV₂₅₄ detection. The metabolite (5)gave a mass of 137 (137.9 MH+)(Mass spectrometer model API300TQMS,Perkin Elmer/Sciex, Toronto, Canada). For Mycobacterium smegmatis (“MSm”or “MSMEG”), macromolecule associated radioactivity was determined byresuspending cells from micro-centrifuged 900 min aliquots (400 μl) inPBS. The cells were ruptured by bead-beating (MiniBeadBeater, BioSpecProducts, Bartlesville, Okla., 3×45sec, 0.1 mm glass beads) andextensively dialyzing the lysates with centricon 10 concentrators(Amicon Inc, Beverly, Mass.) before analysis in 4 ml of EcoscintAscintillation solution.

Example 3. Cloning of EtaA and EtaR

[0075] Genomic DNA from MTb H37Rv was partially digested with Sau3AI(New England BioLabs, Beverly, Mass.) to give fragments of varioussizes. Fragments ranging from 1 Kb to 10 Kb were ligated to pMV206Hyg(Mdluli et al., J Infect Dis 174:1085-90 (1996)) that had beenpreviously linearized with BamHI (New England BioLabs). The ligationmixtures were electroporated into Escherichia coli DH5a (LifeTechnologies, Grand Island, N.Y.) for amplification of the DNA librarywhich was subsequently purified and electroporated into MTb H37Rv. Theresulting transformants were plated on 7H11 (DIFCO) agar plates thatcontained Hygromycin (Life Technologies, 200 μg ml⁻¹) and the indicatedconcentrations of ETA. Five colonies were isolated that had MICs for ETAfrom 2.5 to 5.0 μg/ml (the MIC for wild type MTb is 1.0 μg/ml) (Rist,Adv Tuberc Rec 10:69-126 (1960)).

[0076] EtaA and EtaR were PCR-amplified from H37Rv chromosomal DNA usingthe following primers 5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′(SEQ ID NO:3)and 5′-ATAAGAATGCGGCCGCAACCGTGCTAAAGCTAAACC-3′(SEQ ID NO:4) (Rv3854c,EtaA); 5′-GGGGTACCGCACACTATCGACAC GTAGTAAGC-3′(SEQ ID NO:15) and5′-ATAAGAATGCGGCCGCGCGGTTCTC GCCGTAAATGCT-3′(SEQ ID NO:16) (EtaR) andinserted directionally into KpnI and NotI digested pMH29 (Mdluli et al.,supra).

Example 4. Sequence Analysis of ETA-Resistant Clinical Isolates

[0077] Using the primers described in the previous Example, EtaA was PCRamplified from genomic DNA containing, drug resistant isolate lysates (1ml, bead beaten for 3×45 sec and aqueous diluted 10-fold). EtaA wassequenced in entirety by primer walking for all isolates (SEQWRIGHT Inc,Houston, Tex.) and observed mutations were confirmed on both strands.For the three isolates without mutations in EtaA, EtaR and theintergenic region were also sequenced in entirety without observing anymutations.

Example 5. Synthesis and In Vivo Metabolism of [¹⁴C]-Ethionamide

[0078] We synthesized [¹⁴C]-ETA from 2-ethylpyridine and [¹⁴C]-sodiumcyanide (see Example 1, supra) to study the metabolism of ETA by wholecells of MTh. In the presence of live cells of MTb, ETA is convertedthrough the S-oxide (2) to a single major metabolite (5) as seen by TLCanalysis of sequential time points (FIG. 1A). Metabolites correspondingto the S-oxide (2), nitrile (3), and the amide (4) were identified bycochromatography (TLC and HPLC) with standards synthesized by knownmethods and characterized by ¹H-NMR, ¹³C-NMR and mass spectrometry.These metabolites were produced in small amounts by the cellularoxidation of ETA but they were the dominant products of air oxidation ofETA (compare lanes h and i in FIG. 1A).

[0079] In contrast, metabolite 5 was only produced by live cells of MTband was not seen upon air oxidation of ETA. The thioamide S-oxide 2 wastransiently produced in whole cells and appeared temporally to be aprecursor of metabolite 5 (FIG. 1B). Cold ETA feeding experimentsallowed the isolation of unlabeled metabolite 5 which displayed amolecular mass of 137 by LC-MS (FIG. 1C). We assigned this metabolite as(2-ethyl-pyridin-4-yl)-methanol (5) and confirmed this byco-chromatography (TLC and HPLC) with an authentic synthetic alcoholstandard. The upper HPLC trace in FIG. 1C shows the continuousradio-detector output from a sample corresponding to [1-¹⁴C]ETA that hasbeen air oxidized in media (lane i in FIG. 1A). The lower trace shows asample from MTb metabolism of [1-¹⁴C]ETA after 1.5 hr of exposure (laned in A). The UV₂₅₄ trace of synthetic (2-ethyl-pyridin-4-yl)methanol issuperimposed in gray.

[0080] The production of metabolite (5) from ETA by tuberculosis issurprising as 4-pyridylmethanol is a major metabolite of INH by wholecells of MTb (Youatt, J. Aust J Chem 14:308 (1961); Youatt, J. Aust JExp Biol Med Sci 38:245 (1960); Youatt, J. Aust J Biol Med Sci 40:191(1962)). Like spontaneous oxidation of INH, spontaneous oxidation of ETAfails to produce any trace of the major in vivo metabolite,(2-ethyl-pyridin-4-yl)methanol. INH has been shown to be activated byKatG in vitro to a variety of products including isonicotinic acid,isonicotinamide and isonicotinaldehyde (which in vivo is rapidly reducedto 4-pyridylmethanol) (Johnsson, K. et al., J Am Chem Soc 116:7425(1994)). INH metabolism to 4-pyridylmethanol only occurs indrug-susceptible organisms while drug-resistant organisms no longerproduce this metabolite (Youatt, J., Am Rev Respir Dis 99:729 (1969)).Similarly, we postulate that ETA is activated via the correspondingS-oxide to a sulfinate that can form an analogous aldehyde equivalent(an imine) through a radical intermediate (Paez, O. A. et al., J OrgChem 53:2166 (1988)). (FIG. 5).

Example 6. Identification of a Monooxygenase that Activates ETA.

[0081] To elucidate the enzymatic basis for activation of ETA tometabolite 5 by MTb we selected for ETA resistance in MTb bytransformation of a 1-10 kb insert-containing library of MTh chromosomalDNA in pMV206Hyg (George et al., J. Biol. Chem. 270:27292-8 (1995)).Five colonies were isolated that had MICs for ETA from 2.5 to 5.0 μg/ml(the MIC for wild type MTh is 1.0 μg/ml). Upon restriction analysis thefive independent plasmids were shown to contain the same genomic regionon different overlapping Sau3AI fragments. This cloning was also donewith genomic DNA from a strain reported to be ETA-resistant but the samegenomic locus was obtained with no alterations compared to H37Rv,suggesting that the resistance was not associated with alterations tothis region but simply with its overexpression. The common region to allthe resistance-conferring clones encompassed only one gene (Rv3855,EtaR) that showed broad homology to many TetR family transcriptionalregulators. A 76nt intergenic region separates this putative regulatorfrom a divergently transcribed monooxygenase (Rv3854c, EtaA). One of theisolated library plasmids containing only the etaR gene waselectroporated into MTh and MSm and the resulting MTh transformants grewas a lawn at 2.5 and 5 μg/ml ETA indicating that EtaR was solelyresponsible for ETA resistance. The MSm transformants were able to growat greater than 200 μg/ml ETA, compared to growth of vector controlcontaining MSm at 50 μg/ml.

[0082] Two other monooxygenase/regulator pairs with similar genomicorganization appeared to have high homology in both the regulator andmonooxygenase components to the MTh locus, one from Dienococcusradiodurans (White et al., Science 286:1571-7 (1999)) and the other fromStreptomyces coelicolor (Redenbach et al., Mol Microbiol 21:77-96(1996). This conservation suggested that the effect of regulatorexpression was to modulate production of the adjacent monooxygenase. Tosee if EtaR-mediated repression of EtaA was the cause of ETA resistance,we transformed MTb and MSm with pMH29 plasmid constructs containing etaRand EtaA separately under the control of a strong constituitive promoter(Mdluli et al., supra). Although we could observe resistance with EtaRconstructs in MTh, we were not successful in overexpressing EtaA in MTb,suggesting expression of this enzyme is tightly controlled in thisorganism. MSm overexpressing the putative repressor was found to be ETAresistant with a measured MIC greater than 62.5 μg/ml on solid media(FIG. 2A). Although the recombinant MSm were equally susceptible tokilling with INH, the bacteria overexpressing EtaA were found to behypersensitive to ETA with noticeable growth inhibition at 2.5 μg/ml, alevel comparable to the normal MIC for MTb (FIG. 2A). Qualitativelycomparable results were obtained when these organisms were treated withETA S-oxide (although the absolute MIC for the sulfoxide is lower, EtaRconferred resistance and EtaA conferred hypersensitivity). These resultssuggest that EtaA is directly responsible for thioamide S-oxideoxidative activation and that EtaR modulates expression of this enzyme.

Example 7. Effect of the EtaR Gene

[0083] To link expression of the EtaA activator more directly with ETAmetabolism we examined [¹⁴C]-ETA conversion by whole cells of the MSmtransformants described above over a time-course study as shown in FIG.3. The EtaA overproducing MSm was found to convert ETA to metabolite 5much more quickly than vector control (FIG. 3A). Although the EtaRoverproducing strain did appear to effect this conversion lessefficiently than the control, the result was not dramatic since MSmnormally only weakly activates ETA consistent with this organism'shigher overall MIC for ETA (FIG. 1B). These studies directly correlateETA activation and metabolism with toxicity as measured by MIC. Tounderstand the effect of drug activation we also examined covalentincorporation of [¹⁴C]-ETA into cellular macromolecules by lysingtreated cells and then extensively dialyzing away small molecules. Drugactivation was found to correlate directly with incorporation of labeleddrug into macromolecules (FIG. 2D).

Example 8. Correlation of ETA-Resistance with Resistance to OtherThioamide Drugs

[0084] ETA is only one example of a thiocarbonyl-containingantituberculosis medication approved for clinical use. Among thesecond-line tuberculosis therapeutics there are two other suchmolecules, thiacetazone (11) and thiocarlide (isoxyl) (12) (FIG. 4A)that might be similarly activated by EtaA-catalyzed S-oxidation. Toelucidate the clinical relevance of EtaA-mediated resistance tothiocarbonyl-containing drugs as a class we characterized a set of 14multidrug resistant isolates from patients in Cape Town, South Africa.These isolates were selected on the basis of thiacetazone resistance andthen characterized with respect to ETA resistance. Eleven of fourteen ofthese isolates were found to be ETA cross-resistant. Despite the factthat none of the patients had been treated with thiocarlide, thirteenout of fourteen of the isolates showed thiocarlide cross-resistance.

[0085] To examine at the molecular level the relevance of EtaA-mediatedthiocarbonyl activation for this class of compounds, we PCR-amplifiedand sequenced the EtaA gene from all 14 multidrug-resistant patientisolates. In addition, we examined an in vitro generated ETAmono-resistant strain (ATCC 35830). Eleven of 14 clinical isolates hadamino acid altering mutations in EtaA, as indicated in FIG. 4B.

[0086] EtaA was PCR amplified from chromosomal DNA-containing lysates of1 ml cultures of patient isolates using the primers set forth in Example1, above. EtaA was sequenced in its entirety by primer walking for allisolates and observed mutations were confirmed on both strands. For thethree isolates without mutation in EtaA, EtaR and the intergenic regionwere also sequenced in their entirety without observing any mutations.Eleven of fourteen clinical isolates had amino acid-altering mutationsin EtaA, as indicated in FIG. 3A. The nucleotide change at base 1025 wasfound in two isolates, that at base 1141 in three isolates. Along withthe single nucleotide changes, a 1 nt nucleotide deletion (at base 65)and addition (at base 811) were found. In the ATCC ETA mono-resistantstrain, a nucleotide change at position 557 of EtaA was found. Thepatient isolates in which mutations could not be found (either in EtaA,EtaR or their promoter regions) were subsequently tested and found to befully sensitive to ETA. Thus there is a 100% correspondence betweenmutation in EtaA and ETA cross-resistance among thesethiacetazone-resistant strains.

Example 9. Mechanism of ETA Activation

[0087] INH (6) has been shown to be activated by KatG in vitro to avariety of products including isonicotinic acid, isonicotinamide andisonicotinaldehyde (9) (which in vivo is rapidly reduced to4-pyridylmethanol (10)) (Johnsson, K. & Schultz, P. G., J Am Chem Soc116:7425-68 (1994)). The results support the notion that in vivo INH ismetabolized by oxidation to an acyl diimide (7), then to a diazonium ion(8) or an isonicotinyl radical which may abstract a hydrogen atom from asuitable donor to form isonicotinaldehyde. Similarly, we postulate thatETA is activated via the corresponding S-oxide (2) to a sulfinate thatcan form an analogous aldehyde equivalent (an imine) through a radicalintermediate (FIG. 5). Hydrolysis of this imine could be followed byreduction of the resulting aldehyde to the observed metabolite (5).

[0088] The mechanistic linkage of the activated form of ETA and INHexplains, in part, the observation that they share a final commontarget. The striking observation that both drugs give rise toessentially the same final metabolite upon productive activation of thedrug, further substantiates this common mechanism. Despite thiscommonality, an acyl hydrazide and a thioarnide must undergo verydifferent activation processes by discrete enzymes before they convergeupon an analogous reactive intermediate. The association of KatG withINH activation has been firmly established by a combination of loss ofactivity studies, laboratory-selected drug-resistant mutants,overexpression, and clinically relevant mutations. The results hereestablish that EtaA is the analogous enzyme for the activation of ETAand provide similar evidence based upon genetic manipulation of theenzyme levels and mutations observed in patient isolates.

Example 10. Relationship of EtaA to Other Bacterial Enzymes

[0089] EtaA has two closely related homologs (Rv3083, Rv0565c) encodedwithin the MTb genome that share almost 50% identity to thismonooxygenase (Cole, et al., Nature 393:537-44 (1998)). It is also amember of a family of 14 more loosely related proteins, the majority ofwhich are probable monoxygenases. In addition, MTb has twenty additionalhomologs of Cytochrome P-450 containing oxygenases, the largest numberever identified within a single bacterial genome (Nelson, D. R., ArchBiochem Biophys 369:1-10 (1999)). The reason for this amazing radiationof oxidative enzymes is not clear but they may improve bacterialsurvival in the face of various xenobiotic substances. In this vein, theETA susceptibility of this organism may arise from accidental activationby an enzyme intended to help detoxification.

[0090] Thiacetazone (11) has been widely used as a front-linetherapeutic in Africa and throughout the developing world because it isextremely inexpensive. Although thiocarlide (12) has not been widelyused there is renewed interest in this drug and new analogs. There is animpressive clinical history of cross-resistance among this set of threesecond-line therapies. This cross-resistance suggested a commonmechanism of activation of thiocarbonyl containing molecules that mightallow the simultaneous acquisition of drug resistance to this class oftherapeutic. When we examined patient isolates from Cape Town forcross-resistance to other thioamides or thioureas, we noted that thevast majority of ETA/thiacetazone resistant isolates were alreadyresistant to thiocarlide, despite the fact that these patients werenever treated with this drug.

[0091] The extensive cross-resistance among these compounds predictsmultiple overlapping mechanisms of resistance among clinically usedantituberculars: target associated between INH and ETA, andactivation-associated between ETA, thiacetazone, and thiocarlide. Suchconsiderations complicate appropriate drug therapy for the treatment ofmultidrug-resistant tuberculosis and these results provide an importanttool to help understand and quickly characterize the resistancemechanisms operating in a single patient, which may prove vital to apositive outcome.

[0092] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

What is claimed is:
 1. A method of determining the ability of aMycobacterium tuberculosis bacterium to oxidize a thioamide or athiocarbonyl, said method comprising detecting a mutation in an EtaAgene (SEQ ID NO:1) in said bacterium, wherein detection of the mutationis indicative of decreased ability to oxidize a thioamide or athiocarbonyl.
 2. The method of claim 1, wherein the mutation is aframeshift mutation selected from the group consisting of: a deletion atposition 65, an addition at position 567, and an addition at position811.
 3. The method of claim 1, wherein the mutation is a singlenucleotide polymorphism which causes an amino acid substitution in anamino acid sequence encoded by said EtaA gene compared to an amino acidsequence of SEQ ID NO:2.
 4. The method of claim 3, wherein the singlenucleotide polymorphism causes an amino acid substitution selected fromthe group consisting of: G43C, P51L, D58A, Y84D, T186K, T342K, andA381P.
 5. A method of claim 1 wherein the mutation is detected by (a)amplifying the EtaA gene, or a portion thereof containing the mutation,with a set of primers to provide an amplified product, (b) sequencingthe amplified product to obtain a sequence, and (c) comparing thesequence of the amplified product with the sequence of a wild-type EtaAgene (SEQ ID NO:1) or portion thereof, wherein a difference between thesequence of the amplified product and the sequence of the wild-type EtaAgene or portion thereof indicates the presence of a mutation.
 6. Amethod of claim 5, wherein at least one of said primers is selected fromthe group consisting of: 5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ IDNO:3), 5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′ (SEQ ID NO:4),5′ ATCATCCATCCGCAGCAC 3′ (SEQ ID NO:5); 5′ AAGCTGCAGGTTCAACC 3′ (SEQ IDNO:6); 5′ GCATCGTGACGTGCTTG 3′ (SEQ ID NO:7); 5′ AAGCTGCAGGTTCAACC 3′(SEQ ID NO:8); 5′ TGAACTCAGGTCGCGAAC 3′ (SEQ ID NO:9);5′ AACATCGTCGTGATCGG 3′ (SEQ ID NO:10); 5′ ATTTGTTCCGTTATCCC 3′ (SEQ IDNO:11); 5′ AACCTAGCGTGTACATG 3′ (SEQ ID NO:12); 5′ TCTATTTCCCATCCAAG 3(SEQ ID NO:13); and 5′ GCCATGTCGGCTTGATTG 3′ (SEQ ID NO:14).


7. A method of claim 5, wherein the primers are5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ ID NO:3), and5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′ (SEQ ID NO:4).


8. A method of claim 5, wherein said amplification is by polymerasechain reaction.
 9. A method of claim 1, wherein said mutation isdetected by hybridizing DNA from said bacterium to a test nucleic acidunder stringent conditions.
 10. A method of claim 9, wherein either saidDNA from said bacterium or said test nucleic acid is immobilized on asolid support.
 11. A method of claim 1, wherein said mutation isdetected by (a) subjecting said EtaA gene to digestion by restrictionenzymes, (b) separating the resulting restriction products to form apattern of restriction fragment lengths, and (c) comparing the patternof restriction fragment lengths to a pattern of restriction fragmentlengths formed by subjecting a known EtaA gene to the same restrictionenzymes.
 12. A method of claim 11, wherein said known EtaA gene isselected from the group consisting of (a) a frameshift mutationconsisting of a deletion at position 65, an addition at position 567,and an addition at position 811, and (b) a single nucleotidepolymorphism which causes an amino acid substitution selected from thegroup consisting of: G43C, P51L, D58A, Y84D, T186K, T342K, and A381P.13. A method of claim 1, wherein said mutation is detected byspecifically binding an antibody to a mutated product of the EtaA gene,wherein the specific binding of the antibody to the mutated gene productis indicative of a mutation which inhibits the ability of the bacteriumto oxidize a thioamide.
 14. A method of claim 13, wherein said geneproduct is in, or is isolated from, sputum.
 15. A method of claim 13,wherein detection of said specific binding of said antibody and saidmutated gene product is by ELISA.
 16. A method of claim 1, wherein saidthioamide or thiocarbonyl is selected from the group consisting ofethionamide, thiacetazone, and thiocarlide.
 17. A method of claim 1,wherein said mutation is detected by (a) culturing said bacterium in thepresence of ethionamide; and (b) testing for the presence or absence of(2-ethyl-pyridin-4-yl)methanol, wherein the absence of(2-ethyl-pyridin-4-yl)methanol indicates that the bacterium has amutation which is indicative of decreased ability to oxidize athioamide.
 18. A method of claim 17 wherein the presence or absence of(2-ethyl-pyridin-4-yl)methanol is tested by subjecting a medium in whichthe bacterium is cultured, or the bacterium, to analysis by thin-layerchromatography, high pressure liquid chromatography, or massspectrometry.
 19. A method of claim 17, wherein the ethionamide of step(a) is radioactively labeled.
 20. A method of claim 17, wherein the(2-ethyl-pyridin-4-yl)methanol is radioactively labeled.
 21. A method ofscreening an individual for a Mycobacterium tuberculosis bacteriumresistant to treatment by a thioamide or a thiocarbonyl drug, comprising(a) obtaining a biological sample containing said bacterium from saidindividual, and (b) detecting a mutation in an EtaA gene (SEQ ID NO:1)in said bacterium, wherein detection of the mutation is indicative saidbacterium is resistant to treatment by a thioamide or a thiocarbonyldrug.
 22. A method of claim 21, wherein the mutation is detected by (a)amplifying the EtaA gene with a set of primers to provide an amplifiedproduct, (b) sequencing the amplified product to obtain a sequence, and(c) comparing the sequence of the amplified product with the sequence ofa wild-type EtaA gene (SEQ ID NO:1), wherein a difference between thesequence of the amplified product and the sequence of the wild-type EtaAgene indicates the presence of a mutation.
 23. A method of claim 21,wherein at least one of said primers is selected from the groupconsisting of: 5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ ID NO:3),5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′ (SEQ ID NO:4),5′ ATCATCCATCCGCAGCAC 3′ (SEQ ID NO:5); 5′ AAGCTGCAGGTTCAACC 3′ (SEQ IDNO:6); 5′ GCATCGTGACGTGCTTG 3′ (SEQ ID NO:7); 5′ AAGCTGCAGGTTCAACC 3′(SEQ ID NO:8); 5′ TGAACTCAGGTCGCGAAC 3′ (SEQ ID NO:9);5′ AACATCGTCGTGATCGG 3′ (SEQ ID NO:10); 5′ ATTTGTTCCGTTATCCC 3′ (SEQ IDNO:11); 5′ AACCTAGCGTGTACATG 3′ (SEQ ID NO:12); 5′ TCTATTTCCCATCCAAG 3(SEQ ID NO:13); and 5′ GCCATGTCGGCTTGATTG 3′ (SEQ ID NO:14).


24. A method of claim 21, wherein said primers are5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ ID NO:3) and5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′ (SEQ ID NO:4).


25. A kit for determining the ability of a Mycobacterium tuberculosisbacterium to oxidize a thioamide or a thiocarbonyl, the kit comprising:(a) a container, and (b) primers for amplifying an EtaA gene of saidbacterium or a portion of said EtaA gene containing a mutation affectingthe ability of the bacterium to oxidize a thioamide.
 26. A kit of claim25, wherein at least one of said primers is selected from the groupconsisting of: 5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ ID NO:3),5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′ (SEQ ID NO:4),5′ ATCATCCATCCGCAGCAC 3′ (SEQ ID NO:5); 5′ AAGCTGCAGGTTCAACC 3′ (SEQ IDNO:6); 5′ GCATCGTGACGTGCTTG 3′ (SEQ ID NO:7); 5′ AAGCTGCAGGTTCAACC 3′(SEQ ID NO:8); 5′ TGAACTCAGGTCGCGAAC 3′ (SEQ ID NO:9);5′ AACATCGTCGTGATCGG 3′ (SEQ ID NO:10); 5′ ATTTGTTCCGTTATCCC 3′ (SEQ IDNO:11); 5′ AACCTAGCGTGTACATG 3′ (SEQ ID NO:12); 5′ TCTATTTCCCATCCAAG 3(SEQ ID NO:13); and 5′ GCCATGTCGGCTTGATTG 3′ (SEQ ID NO:14).


27. A kit of claim 25, wherein the primers are5′-GGGGTACCGACATTACGTTGATAGCGTGGA-3′ (SEQ ID NO:3), and5′-ATAAGAATGCGGCCGCAACCGTCGCTAAAGCTAAACC-3′ (SEQ ID NO:4).


28. A kit of claim 25, further comprising a mutated EtaA gene for use asa positive control.
 29. A kit of claim 28, wherein said mutated EtaAgene is selected from the group consisting of (a) a frameshift mutationconsisting of a deletion at position 65, an addition at position 567,and an addition at position 811, and (b) a single nucleotidepolymorphism which causes an amino acid substitution selected from thegroup consisting of: G43C, P51L, D58A, Y84D, T186K, T342K, and A381P.30. A kit for determining the ability of a Mycobacterium tuberculosisbacterium to oxidize a thioamide, the kit comprising: (a) a container,and (b) (2-ethyl-pyridin-4-yl)methanol.
 31. A kit for determining theability of a Mycobacterium tuberculosis bacterium to oxidize athioamide, the kit comprising: (a) a container, and (b) radiolabeledethioamide.
 32. A kit for determining the ability of a Mycobacteriumtuberculosis bacterium to oxidize a thioamide or thiocarbonyl, the kitcomprising: (a) a container, and (b) an antibody which specificallybinds to a product of a EtaA gene selected from the group consisting ofa wild-type EtaA gene (SEQ ID NO:1) and a mutated EtaA gene.
 33. A kitfor determining the ability of a Mycobacterium tuberculosis bacterium tooxidize a thioamide, the kit comprising: (a) a container, and (b) anantibody which specifically binds to (2-ethyl-pyridin-4-yl)methanol.